16] Enzyme probes in Vitro

Abstract

This chapter discusses the structure of DNA structure using enzymatic probes. The most widely used enzymatic probes for DNA structure in vitro are endonucleases with little or no sequence specificity, such as the family of zinc-dependent single-strand-specific endonucleases that comprise S1 nuclease, P1 nuclease, mung bean nuclease, and others. In general, double-stranded DNA is cleaved by these enzymes at low rates, whereas single strands are digested rapidly. If structural transitions in the DNA lead to unpairing of complementary bases, the nuclease then cuts at this site. Because this provides a positive signal for a structural transition, nucleases have been extensively used to monitor conformational parameters in nucleic acids. S1 nuclease from Aspergillus oryzae has been widely used to probe structural transitions in DNA. It has a pH optimum in the acidic range (pH 5 and below), which makes it a good probe for the investigation of protonated structures but restricts its use under physiological conditions. P1 nuclease from Penicillium citrium acts in many ways similarly to S1 nuclease. An important difference is its ability to cut DNA well at neutral pH values, although its pH optimum is acidic. The products of P1 nuclease action are oligonucleotides with 5´-phosphates. Mung bean nuclease is related to the S1 and P1 nucleases. The enzyme is highly sensitive to small variations in DNA structure and converts single-stranded DNA to mono- or oligo-nucleotides with 5´-phosphates.